Physiologically acceptable aqueous solutions and methods for their use

ABSTRACT

Physiologically acceptable aqueous solutions and methods for their use are provided. The subject solutions comprise: electrolytes; a dynamic buffering system and an oncotic agent; and do not comprise a conventional biological buffer. The subject solutions find use in a variety of applications, particularly in those applications where at least a portion of a host&#39;s blood volume is replaced with a blood substitute.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/886,921 filed Jul. 2, 1997 and now issued as U.S. Pat. No. 5,945,272;which is a continuation of application Ser. No. 08/780,974 filed on Jan.9, 1997, now abandoned; which application is a continuation ofapplication Ser. No. 08/364,699 filed Dec. 8, 1994, now abandoned; whichapplication is a continuation-in-part of application Ser. No. 08/253,384filed Jun. 3, 1994 and now issued as U.S. Pat. No. 5,702,880; whichapplication is a continuation-in-part of application Ser. No. 08/133,527filed Oct. 7, 1993 and now abandoned; which application is acontinuation-in-part of application Ser. No. 08/071,533, filed Jun. 4,1993 and now issued as U.S. Pat. No. 5,407,428; the disclosures of whichapplications are herein incorporated by reference and to whichapplications we claim priority under 35 U.S.C. § 120.

INTRODUCTION

1. Technical Field

The technical field of this invention is plasma substitute solutions.

2. Background of the Invention

Physiologically acceptable solutions find use in a variety of differentapplications in the medical, biomedical research and related fields. Forexample, physiologically acceptable solutions find use as plasmasubstitutes in surgical applications which require the replacement ofsignificant amounts of blood plasma volume. Such applications includetreatments for blood lost during surgery or trauma, or when a tissue,organ, group of organs or an entire subject needs to be maintained at ahypothermic or frozen state. Such applications also include applicationsin which a patient's blood is flowed through an external device, such asa cardiopulmonary bypass machine, where the extra circulatory volumespace resulting from attachment of the patient's circulatory system tothe device must be filled with a compatible blood substitute, i.e. bloodvolume expander.

Physiologically acceptable solutions suitable for use as plasmaexpanders/substitutes must be able to mix freely with blood withoutunacceptably compromising its components, such as creating precipitateswhich significantly block flow in small vessels, destroying anunacceptable portion of its formed elements (cells, platelets),introducing agents or creating water, ionic or molecular imbalancesdestructive to body cells and tissues, or causing harmful physiologicactivities such as inappropriate acceleration or inhibition ofheartbeat, nerve conduction or muscle contraction, and the like.

The first plasma substitute solutions employed were derived frommammalian blood. Although such solutions have been used with success,because such solutions are derived from natural blood, they can containvarious pathogenic substances, such as viral pathogens such as HIV,Hepatitis B, and other pathogens, e.g. prions such as those associatedwith Cruetzfeldt-Jakob disease, and the like. As such, use of bloodsubstituted and plasma substitute solutions derived from natural bloodare not free of complication.

As such, a variety of synthetic blood and plasma substitute solutionshave been developed which are prepared from non-blood derivedcomponents. Although synthetic plasma like solutions have foundincreasing use in a variety of applications, no single solution hasproved suitable for use in all potential applications.

Accordingly, there is continued interest in the development of newphysiologically acceptable aqueous solutions that are suitable for useas plasma substitutes. Of particular interest is the development ofsolutions that are suitable for use in hypothermic surgicalapplications, such as cardiac surgery and the like. Also of interest isthe development of solutions that are terminally heat sterilizable.

Relevant Literature

Various physiologically acceptable solutions, particularly bloodsubstitute solutions, and methods for their use are described in U.S.Pat. Nos. : RE 34,077; 3,937,821; 4,001,401; 4,061,736; 4,216,205;4,663,166; 4,812,310; 4,908,350; 4,923,442; 4,927,806; 5,082,831;5,084,377; 5,130,230; 5,171,526; 5,210,083; 5,274,001; 5,374,624; and5,407,428.

Additional references describing physiologically acceptable solutions,including blood substitute solutions include: Bishop et al.,Transplantation (1978) 25:235-239; Messmer et al., Characteristics,Effects and Side-Effects of Plasma Substitutes, pp 51-70; Rosenberg,Proc.12th Congr. Int. Soc. Blood Transf.(1969); Spahn, Anesth. Anaig.(1994) 78:1000-1021; Biomedical Advances In Aging (1990)(Plenum Press)Chapter 19; Wagner et al., Clin. Pharm. (1993) 12:335; ATCC Catalogue ofBacteria & Bacteriophages (1992) p 486; and 06-3874-R8-Rev. May (1987)Abbott Laboratories, North Chicago, Ill. 60064, USA.

Additional references describing various applications of such solutions,including hypothermic applications, include: Bailes et al., Cryobiology(1990) 27:615-696(pp 622-623); Belzer et al., Transplantation (1985)39:118-121; Collins, Transplantation Proceedings (1977) 9:1529; Fischeret al., Transplantation (1985) 39:122; Kallerhoff et al.,Transplantation (1985) 39:485; Leavitt et al., FASB J. (1990) 4: A963;Ross et al., Transplantation (1976) 21:498; Segall et al. FASB J. (1991)5:A396; Smith, Proc. Royal Soc. (1956) 145: 395; Waitz et al., FASB J.(1991) 5.

Lehninger, Biochemistry (2^(nd) Ed., 1975), pp 829ff provides a reviewof blood and its constituents.

SUMMARY OF THE INVENTION

Physiologically acceptable aqueous solutions and methods for their useare provided. The subject solutions comprise: electrolytes; a dynamicbuffering system and an oncotic agent; where the solutions do notcomprise a conventional biological buffer. The solutions find use in avariety of applications, particularly in applications in which at leastof portion of a host's blood volume is replaced with a blood substitutesolution.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Physiologically acceptable aqueous solutions and methods for their useare provided. The subject solutions include: electrolytes; a dynamicbuffering system and oncotic agents; where the solutions may furtheroptionally include at least one of a sugar and bicarbonate and willinclude at least one of magnesium or sugar. The solutions may be used ina variety of applications and are particularly suited for use inapplications where at least a portion of a host's blood is replaced witha substitute solution. In further describing the invention, the aqueoussolutions themselves will be described first in greater detail followedby a discussion of various representative applications in which thesolutions find use.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

The aqueous solutions of the subject invention are physiologicallyacceptable, by which is meant that the solutions may be introduced intothe vasculature of a host without inherently causing a toxic reaction.The solutions will have a pH ranging from about 4 to 10, usually fromabout 4.5 to 9 and more usually from about 5 to 8.5.

The solutions will comprise a plurality of electrolytes, including:sodium ion, chloride ion, potassium ion and calcium ion, and optionallymagnesium ion. The sodium ion concentration of the solutions will rangefrom about 70 to 160, usually from about 110to 150, and in someembodiments from 130 to 150 mM. The concentration of chloride ion in thesolution will range from about 70 to 170, usually from about 80 to 160,more usually from about 100 to 135 and in some embodiments from about110 to 125 mM. The concentration of potassium ion will range from thephysiological to subphysiological, where by “physiological” is meantfrom about 3.5 to 5, usually from about 4 to 5 mM, and by“subphysiological” is meant from about 0 to 3.5, usually from about 2 to3 mM, where in many embodiments of the invention, the amount ofpotassium ion will range from about 1 to 5, usually from about 2-3 mM,where in certain embodiments, the amount of potassium ion may be higherthan 5 mM and range as high as about 5.5 mM or higher, but will usuallynot exceed about 5.5. mM. The solutions will also comprise calcium ionin an amount ranging from about 0.5 to 6.0 mM, and in many embodimentswill range from about 0.5 to 4.0, usually from about 2.0 to 2.5 mM, butin certain embodiments will range from about 4.0 to 6.0, usually fromabout 4.5 to 6.0 mM. Optionally, the solutions may further comprisemagnesium. When present, the magnesium ion will range from about 0 to 10mM, usually from about 0.3 to 3.0 and more usually from about 0.3 to0.45 mM.

In certain embodiments, the subject solutions will comprise elevatedlevels of both potassium and magnesium. By elevated levels is meant apotassium ion concentration in an amount ranging from about 50 mM to 3.0M, usually from about 200 mM to 2.5 M, and more usually from about 1.0to 2.5 M, and a magnesium ion concentration of from about 40 mM to 1.0M, usually from about 0.1 to 0.9 M and more usually from about 0.3 to0.7 M.

Also of interest are solutions which comprise elevated levels ofpotassium and a magnesium electrolytes (known as “superchargersolutions”). By elevated levels is meant a potassium ion concentrationin an amount ranging from about 50 mM to 3.0 M, usually from about 200mM to 2.5 M, and more usually from about 1.0 to 2.5 M, and a magnesiumion concentration of from about 40 mM to 1.0 M, usually from about 0.1to 0.9 M and more usually from about 0.3 to 0.7 M. Theses solutions mayfurther comprise, in certain embodiments, bicarbonate, where thebicarbonate will be present in amounts ranging from about 0.1 to 40 mM,usually from about 0.5 to 30 mM and more usually from about 1 to 10 mM.

The solutions also comprise a dynamic buffering system, where the termdynamic buffering system is used to refer to one, or more reagents thatwork in combination to keep the pH of the solution in a certain range inan in vivo environment. Preferably, the reagent members off the dynamicbuffering system are normal biological components that maintain in vivobiological pH. The dynamic buffering system concept rests on thediscovery by the inventors that compounds with no intrinsic bufferingcapacity in the biological range, such as lactate, acetate, or gluconatewhich are capable of being metabolized in vivo, act with other solutioncomponents to maintain a biologically appropriate pH in an animal, evenat hypothermic temperatures and at essentially bloodless conditions. Thedynamic buffering system of the present invention depends in part onoxygenation and removal of carbon dioxide (CO₂). The dynamic buffer ofthe invention has no or substantially no ability to act as a bufferoutside of a biological system, i e., a dynamic buffer maintains pH inthe biological range in vivo but not in a cell free environment.

A critical component of the dynamic buffering system of the invention isa carboxylic acid, salt or ester thereof. By a carboxylic acid, salt orester thereof is meant a compound having the generaI structural formulaRCOOX, where R is an alkyl, alkenyl, or aryl, branched or straightchained, containing 1 to 30 carbons which carbons may be substituted,and preferably one of the carbon chains that compose the carbon chain oflactate, acetate, gluconate, citrate, pyruvate, or other biologicalmetabolites; and X is hydrogen or sodium or other biologicallycompatible ion substituent which can associate at the oxygen position.

Optionally, the dynamic buffering system may further comprise a sourceof bicarbonate, usually sodium bicarbonate (NaHCO₃). When present, theconcentration of NaHCO₃ will range from about 0.1 mM to 40 mM, usuallyfrom about 0.5 mM to 30 mM, and more usually from about 1 mM to 10 mM.

The solution of the present invention does not include a conventionalbiological buffer. By “conventional buffer” is meant a compound which insolution, in vitro, maintains pH at a particular range. By “conventionalbiological buffer” is meant a compound which in a cell-free systemmaintains pH in the biological range of 7-8. Examples of conventionalbiological buffers includeN-2-Hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES),3-(N-Morpholino) propanesulfonic acid (MOPS),2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid(TES), 3-[N-tris(Hydroxy-methyl)ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid (EPPS),Tris[hydroxymethyl]-aminomethane (THAM), and Tris[hydroxymethyl]methylaminomethane (TRIS). Conventional biological buffers have a pK in thephysiological range and function most efficiently in this range.Therefore, these buffers function independently of normal biologicalprocesses and are most potent in cell-free systems.

The absence of a conventional biological buffer in the solution of theinvention confers several important medical advantages. For example,lower concentrations of buffers consisting of normal biologicalcomponents are required to maintain in vivo pH, compared to conventionalbiological buffers. Conventional biological buffers may also posetoxicity problems. Further, the absence of a biological buffer allowsthe solution to be terminally heat sterilized. Generally, medicalsolutions are preferred to be terminally heat sterilized prior to use ina patient. The term “terminally heat sterilized” or “heat sterilized” asused herein refers to the process involving heating a solution to about120° C. for 15 minutes under pressure, i.e., maintaining heat andpressure conditions for a period of time sufficient to kill all orsubstantially all bacteria and inactivate all or substantially allviruses in the solution. This procedure is normally performed in anautoclave, and is also known as “autoclaving”. The purpose of heatsterilization is to kill possible infectious agents present in thesolution. Infectious agents are known to tolerate temperatures up to100° C. It is generally considered by the art that heating a solutionunder pressure to 120° C. for about 15 minutes is sufficient to insuresterility.

The solutions will also comprise an oncotic agent. The oncotic agent iscomprised of molecules whose size is sufficient to prevent its loss fromthe circulation by readily traversing the fenestrations of the capillarybed into the interstitial spaces of the tissues of the body. As a group,oncotic agents are exemplified by blood plasma expanders. Compoundsfinding use as oncotic agents in the subject invention may be natural orsynthetic, and will usually be polymeric compositions having an averagemolecular weight of at least about 40,000, usually at least about100,000 and more usually at least about 200,000, where oncotic agentshaving a molecular weight of 300,000 or higher may find use. Examples ofoncotic agents suitable for use in the solution of the present inventioninclude proteinaceous compounds, such as albumin, e.g. human serumalbumin, and cross-linked or high molecular weight hemoglobin,polysaccharides such as glucan polymers, and the like; organic polymers,e.g. PVP, PEG, etc.; and the like; where non-antigenic polysaccharidesare preferred;

Polysaccharides that find use as oncotic agents in the subject solutionsinclude hydroxyethyl starches, hydroxymethyl alpha (1→4) or (1→6)polymers, D-glucose polymers, e.g. dextrans having an alpha (1→6)linkage, cyclodextrins, hydroxypropylstarches, hydroxyacetylstarches,and the like.

Hydroxyethyl starches are of particular interest for certain embodimentsof the subject invention. The average molecular weight of hydroxyethylstarches finding use in the subject invention may range from 10,000 d to1,000,000 d or higher, where the molecular weight will typically rangefrom about 40,000 d to 1,000,000 d, usually from about 100,000 to900,000, and more usually from about 200,000 to 800,000. Preferred arecompositions in which the average molecular weight of the hydroxyethylstarch oncotic agent ranges from about 50,000 d to 1,000,000 d, usuallyfrom about 100,000 to 900,000 and more usually from about 200,000 to800,000. The degree of substitution will range from about 4 to 10, wherein certain embodiments, the degree of substitution will range from 7 to10, in other embodiments will range from 4 to 5, and in otherembodiments will range from 6 to 7. Therefore, one class of preferredsolutions will comprise a hydroxyethyl starch with between about 6 and 7hydroxyethyl groups for every 10 glucose units. Another class ofpreferred solutions will comprise between about 4 and 5 hydroxyethylgroups for every 10 glucose units. Yet another class of preferredsolutions will comprise between about 7 and 8 hydroxyethyl groups forevery 10 glucose units.

A particularly preferred oncotic agent is Hetastarch (McGaw, Inc.), anartificial colloid derived from a waxy starch composed almost entirelyof amylopectin with hydroxyethyl ether groups introduced into the alpha(1→4) linked glucose units and having a molar substitution of about 0.7hydroxyethyl groups/glucose unit. The colloid properties of a 6%solution (wt/wt) of Hetastarch approximates that of human serum albumin.

Another particularly preferred oncotic agent is Pentastarch, which has amolar substitution of about 0.45 hydroxyethyl groups/glucose unit and anaverage molecular weight range (as measured by the HPSEC method asreported in PDR 1996) of from about 150,000 to 350,000 d, with 80%between 10,000 and 2,000,000 d.

Another particularly preferred oncotic agent is “Hexastarch,” which hasa molar substitution of about 0.64 hydroxyethylgroups/glucose unit andan average molecular weight of about 220,000.

In certain embodiments, the hydroxyethyl starch will be a selectfraction of the initial hydroxyethyl starch source, particularly aselect size fraction, where generally the fraction will be at least oneof the fraction having an average molecular weight of less than about1,000,000 daltons or the fraction having an average molecular weight ofgreater than about 50,000 daltons. Conventional fractionation means maybe used to prepare such fractions.

The concentration of oncotic agent in the solution is sufficient toachieve (when taken together with chloride salts of sodium, calcium andmagnesium, organic ion from the organic salt of sodium and hexose sugardiscussed above) colloid osmotic pressure approximating that of normalhuman serum, about 28 mm Hg. Generally, the amount of oncotic agent inthe solution will range from about 0.5 to 30, usually from about 1 to 25and more usually from about 2 to 8%. Where the oncotic agent is ahydroxyethyl starch, the amount present in the solution will range fromabout 1 to 30, usually from about 2 to 15 and more usually from about 4to 8%.

In one aspect of the invention, the solution contains two or moreoncotic agents with differential clearance rates. The solutions of thepresent invention having two or more oncotic agents with differentialclearance rates provide additional advantages in restoring blood oncoticpressure in a hypovolemic subject over an extended period of time, whileencouraging the subject's own production of plasma proteins. Artificialoncotic agents with relatively slow clearance rates include highmolecular weight Hetastarch (molecular weight 300,000-1,000,000) anddextran 70, measured to have intravascular persistence rates of 6 hours(Messmer (1989) Bodensee Symposium on Microcirculation (Hammersen &Messmer, eds.), Karger, N.Y., pg. 59). Artificial oncotic agents withrelatively fast clearance rates include low and medium molecular weightHetastarch (average molecular weight 40,000-200,000) and dextran 40,having intravascular persistence rates of 2-3 hours (Messmer (1989)supra).

The solution may further comprise one or more different optional agentswhich may be included in the solution to make the solution suited for aparticular application. One optional agent that may be included, andusually is included, is sugar. The sugar will generally be a hexosesugar, such as glucose, fectose and galactose, of which glucose ispreferred. In the preferred embodiment of the invention nutritive hexosesugars are used and a mixture of sugars can be used. The sugar istypically, though not necessarily, present in the solution in aphysiological amount. By the term “physiological amount” or“physiological levels” is meant the concentration of sugar is in a rangebetween 2 mM and 50 mM with concentration of glucose of 5 mM beingpreferred. At times, it is desirable to increase the concentration ofhexose sugar in order to lower fluid retention in the tissues of asubject. Thus the range of hexose sugar may be expanded up to about 50mM or even above, but usually not above 60 and more usually not above 55mM, if necessary to prevent or limit edema in the subject undertreatment, except where the agent is present as a cryoprotective agent.

The solutions of the present invention may include a blood clottingfactor able to accelerate or promote the formation of a blood clot.Preferred blood clotting factors for use in the solution of theinvention include vitamin K, Factors I, II, V, VII, VIII, VIIIC, IX, X,XI, XII, XIII, protein C, von Willebrand factor, Fitzgerald factor,Fletcher factor, and a proteinase inhibitor. The concentration of theblood clotting factor is determined by one skilled in the art dependingon the specific circumstances of treatment. For example, generally whenvitamin K is administered, its concentration will be sufficient todeliver 5-10 mg to the patient.

The solutions of the present invention may include an oxygen-carryingcomponent in a concentration sufficiently low so as not to be toxic tothe subject. The oxygen carrying component will usually be present in asufficient amount to deliver enhanced oxygen to the tissues of a subjectwithout resulting in toxicity to the subject. A “sufficient amount” ofan oxygen-carrying component is an amount allowing a resting subjectwith an unimpaired circulation and physiology to survive and recoverfrom trauma, illness or injury. In normal humans at normal bodytemperature, this is at least 5-6 ml O₂/100 ml of intravascular fluid.Oxygen-carrying components include hemoglobin extracted from human andnon-human sources, recombinant hemoglobin, hemocyanin, chlorocruorin andhemerythrin, and other naturally occurring respiratory pigmentsextracted from natural sources or made by recombinant DNA or in vitromethods. These compounds may be modified by a number of means known tothe art, including by chemical crosslinking or covalent bonding topolyethylene glycol group(s). When the oxygen-carrying component ishemoglobin, it is preferably present in the concentration range ofbetween about 20-200 g/l.

The solutions may further comprise one or more cryoprotective agents,where by cryoprotective agent is meant any agent that preserves thestructural integrity of tissue under hypothermic, e.g. sub-zero,conditions, where in certain embodiments the cryoprotective agent willbe an agent that disrupts, at least to a partial extent, the orderedcrystal arrangement of water molecules in a manner such that thefreezing point of the aqueous solution comprising the cryoprotectiveagent is lowered as compared to the freezing point of an analogoussolution that does not comprise a cryoprotective agent. Cryoprotectiveagents of interest include: alcohols, particularly low molecular weightaliphatic alcohols, usually C1 to C6 alcohols, more usually C1 to C4alcohols, such as methanol, ethanol, and the like; polyols, includinglinear, branched and cyclic polyols, usually low molecular weightaliphatic polyols, including diols, triols, and other polyols, such assugars (described in greater detail below) where polyols of particularinterest include diols, such as ethylenediol, propanediol, butanediol,triols, e.g. glycerol, and the like; sugars, including erythrose,threose, ribose, arabinose,-xylose, lyxose, allose, atrose, glucose,mannose, gulose, idose, galactose, talose, erythrulose, ribulose,xylulose, psicose, fructose, sorbose, tagatose and disaccharides, e.g.sucrose, lactose and maltose, where glucose is particularly preferred;other agents such as timethylamine, trimethylamine oxide (TMAO), DMSO,urea, formamide, dimethylformamide and the like; clathrates, siliconcomprising agents, such as silanes and the like, fluorocarbon compoundsand derivatives thereof; etc; where the cryoprotective agent may beforced into solution by pressure and/or a suitable surfactant agent maybe employed, where such surfactant agents are known to those of skill inthe art. Such agents will typically be present in amounts sufficient toprovide the desired cryoprotective effect, where the particular amountof the agent will depend on the particular agent employed. When theagent is a polyol, e.g. a diol, it will generally be present in amountsranging from about 0.2 to 1 M or 0 to 30%. With respect to propanediol,in particular a range of 0.2 M to 0.6 M is preferred and a concentrationof about 0.4 M propanediol is most preferred. 1,2 propanediol ispreferred as the adduct to the solution used for low temperature organand donor preservation according to the invention, although 1,3propanediol may be used. For TMAO, TMAO will be present in the solutionin a final concentration in a range between 0.2 M and 7M. When glycerolis employed, it will be present in a concentration ranging from about 0to 40%, usually from about 5 to 30%, and more usually 5 to 20%. WhenDMSO is employed, it will be present in amounts ranging from about 0 to40%, usually from about 5 to 30%, and more usually from about 5 to 20%.When a sugar is employed (particularly glucose), the sugar rangesbetween about 0.6 M to about 1.4 M, with 1.0 M being preferred forcertain embodiments.

In one class of preferred embodiments, the solutions of the subjectinvention will comprise at least two of magnesium ion, a sugar such asglucose, and a medium to high molecular weight hydroxyethyl starch, andmay comprise both components.

The following solution embodiments are of particular interest:

Solution A High Molecular Weight Hetastarch 1 to 10% (average molecularwt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40mM glucose 0-50 mM Solution B High Molecular Weight Hetastarch 1 to 10%(average molecular wt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++0-10 mM lactate 1-40 mM glucose 0-50 mM bicarbonate 5-10 mMCryoprotective Solutions I. High Molecular Weight Hetastarch 1 to 10%(average molecular wt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++0-10 mM lactate 1-40 mM glucose 0-50 mM bicarbonate 5-10 mM glycerol10-20% II. High Molecular Weight Hetastarch 1 to 10% (average molecularwt. of 350,000-900,000) Ca++ 1-6 mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40mM bicarbonate 5-10 mM glycerol 10-20% III. High Molecular WeightHetastarch 1 to 10% (average molecular wt. of 350,000-900,000) Ca++ 1-6mM K+ 1-5 mM Mg++ 0-10 mM lactate 1-40 mM glucose 0-50 mM bicarbonate5-10 mM glycerol 5-15% DMSO 5-15%

In preparing the subject solutions, the various constituents may becombined at substantially the same time, or added sequentially, as maybe convenient. The solutions may be terminally heat sterilized asdescribed above. As also described above, the solutions may furthercomprise agents that should not be terminally heat sterilized, such as asource of bicarbonate, where the bicarbonate participates in the dynamicbuffering system. In such instances, the sodium bicarbonate will beadded as a sterile solution to a pre-autoclaved “base solution.”Similarly, when it is desirable to add a blood clotting factor oroxygen-carrying component, the blood clotting factor or oxygen-carryingcomponent is added as a sterile solution to the autoclaved basesolution.

For purposes of description of the invention, the mixture according tothe invention has been discussed and will continue to be discussed interms of an aqueous solution. From the following description of theinvention, it is expected that one of ordinary skill in the art would beenabled to provide the mixture as a dry mixture and make the adjustmentsto amounts of sodium chloride and organic salt of sodium as necessary toaccommodate the amounts of sodium chloride found in normal salinesolution, which may be used as a diluent for the dry mixture accordingto the invention.

The subject solutions find use in a variety of different applications.The subject solutions find particular use in applications where it isdesired to replace at least a portion of a host's (or tissue or organthereof) circulating blood volume with a substitute solution, where suchapplications include: surgical procedures, including proceduresinvolving a reduction in the temperature of a host from the host'snormal body temperature; as a blood substitute; to maintainphysiological integrity following death; as a cold preservation agentfor tissue or organ; in regional chemoperfusion; and the like.

The solution may be used as a circulating solution in conjunction withoxygen or hyperbaric oxygen at normal body temperatures or duringprocedures when the subject's body temperature is reduced significantlybelow the subject's normal temperature. For example, during surgicalprocedures and in cadaver organ donation at low temperatures, thesubject's blood may be replaced with the cold circulating solution ofthe invention, where the solution may be circulated for a time toperfuse and maintain the subject and its organs intact during theprocedure.

The solution of the present invention may be administered intravenouslyor intra arterially to a euthermic subject which is placed in apressurized atmosphere of increased oxygen concentration up to 100%oxygen or to such a subject undergoing a procedure during which thesubject's body temperature is reduced significantly below the subject'snormal temperature whether or not hyperbaric oxygen is used. While thesolution is being administered to and circulated through the subject,various agents such as cardioplegic agents may be administered eitherdirectly into the subject's circulatory system, administered directly tothe subject's myocardium, or added to the circulating solution of thepresent invention. These components are added to achieve desiredphysiological effects such as maintaining regular cardiac contractileactivity, stopping cardiac fibrillation or completely inhibitingcontractile activity of the myocardium or heart muscle.

Cardioplegic agents are materials that cause myocardial contraction tocease and include anesthetics such as lidocaine, procaine and novocaineand monovalent cations such as potassium ion in concentrationssufficient to achieve myocardial contractile inhibition. Concentrationsof potassium ion sufficient to achieve this effect are generally inexcess of 15 mM, and magnesium may also be present in amounts in excessof about 0.5 mM.

During revival of a subject (after a period of subnormal temperature orcryogenic maintenance using the solution according to the invention tomaintain the subject) the subject may be reinfused with a mixture of thesolution according to the invention along with blood retained from thesubject or obtained from blood donors. As the subject is warmed, wholeblood is infused until the subject achieves an acceptable hematocrit,generally exceeding hematocrits of about 20%. When an acceptablehematocrit is achieved, perfusion is discontinued and the subject isrevived after closure of surgical wounds using conventional procedures.

In general, the solution according to the invention is administeredusing an intravenous line (when the subject is at normal temperature) orto a chilled subject using a pumped circulating device such as acentrifugal pump, roller pump, peristaltic pump or other known andavailable circulatory pump. The circulating device is connected to thesubject via cannulae inserted surgically into appropriate veins andarteries. When the solution is administered to a chilled subject, it isgenerally administered via an arterial cannula and removed from thesubject via a venous cannula and discarded, stored or circulated.

The solution may be used in a variety of surgical settings andprocedures. It may be useful in delicate neurosurgery where clearsurgical fields are imperative and reduced central nervous systemactivity may be desirable and achieved by performing the procedure on apatient whose core temperature and/or cerebral temperature has beensubstantially reduced.

The solution may be used to maintain a subject (which has lost asignificant amount of blood, e.g. 20% to 98% of its blood) at normalbody temperatures in a pressurized environment at increased oxygenconcentration above atmospheric oxygen tension up to 100% oxygen. Thesubject is maintained in a high oxygen concentration, eithercontinuously or periodically, until enough blood components can besynthesized by the subject to support life at atmospheric pressure andoxygen concentration. The solution according to the invention may beused to maintain a subject at temperatures lower than normal bodytemperature and at a reduced rate of metabolism after traumatic lifethreatening injury until appropriate supportive or corrective surgicalprocedures can be performed. In addition the solution may be used tomaintain a patient having a rare blood or tissue type until anappropriate matching donor can be found and replacement blood units orother organ can be obtained.

Surprisingly it has been discovered that it is possible to replacesubstantially all of a mammalian subject's circulating blood with thesolution according to the invention and to maintain the subject alivewithout reinfusing blood into the subject. Substantially all of amammalian subject's circulating blood is considered to be replaced whenthe subject's hematocrit drops below 10%. Hematocrit may be lower than10% if O₂ is provided to the subject, or substantially lower than 10% ina hyperbaric O₂ chamber. The solution according to the invention can ofcourse be used to maintain a subject having a hematocrit in excess of10%.

The procedure for replacing substantially all of a mammalian subject'scirculating blood may be carried out with the mammalian subject's bodytemperature being maintained at its substantially normal temperature. Inaddition the procedure may be carried out with cooling of the subjectand reduction of the mammalian subject's body temperature below that ofits normal temperature. Cooling may be accomplished by chilling thesubject in an ice bath, ice-salt slurry, or cooling blanket.

The subject may be further cooled by chilling the solution according tothe invention prior to perfusing the subject with the solution.

In the procedure according to the invention for replacing substantiallyall of a mammalian subject's circulating blood, it is preferred that thesubject is chilled and perfused with the solution, using an arterialcatheter to deliver the solution to the subject's circulatory system anda venous catheter to remove blood and the perfusate from the subject.Substantially all of the subject's circulating blood is removed in thismanner as determined by measurement of the hematocrit of the effluentfrom the venous catheter. When substantially all of the subject'scirculating blood is removed, perfusion may be stopped.

In addition, the procedure for replacing substantially all of thesubject's blood may be carried out with the aid of hyperbaric O₂. Thesubject is placed in a hyperbaric chamber pressurized with oxygen atconcentrations exceeding 20%, preferably 100% oxygen. The pressure ofthe hyperbaric chamber is maintained during most of the procedure in arange between 0.5 pounds per square inch over atmospheric pressure topressures up to about twice atmospheric pressure. In one embodiment, theprocedure is performed with the subject in a hyperbaric chamber athyperbaric pressures of about 0.07 to about 2 atmospheres over ambientpressure (0.5-30 pounds per square inch [psi]) with 100% oxygen. Ifnecessary, the pressure of the hyperbaric chamber may be reduced toatmospheric pressure during wound closure. The subject is subsequentlymaintained at hyperbaric pressure at high oxygen concentration. Thepressure is gradually reduced to a lower pressure but one stillhyperbaric. Preferably the pressure is maintained below 10 psi to about5 psi for a number of hours to several days.

Subsequently, the pressure is again gradually lowered below 1 psi andpreferably to about 0.5 psi and is maintained at this pressure for anadditional period of time up to a day or more.

The solution may also be used to maintain the physiological integrity ofan organ donor subject immediately after the occurrence of brain death.The subject can be chilled, the subject's blood removed and replacedwith a circulating solution maintained below 37° C., or whilecirculating cold solution according to the invention. Through this useof the solution, ischemia of vital organs can be minimized. Bycirculating cold solution according to the invention through thesubject's circulatory system at low temperature with or without placingthe subject in a hyperbaric oxygen chamber, vital organs can bemaintained for longer periods of time, thus maximizing the number oforgans that can be effectively used from one donor for potentialtransplant recipients.

In another aspect of the invention, it has been discovered that by usingcertain adducts, particularly propanediol and high concentration glucoseto augment the solution, it may be possible to reduce the temperature ofdonor organs, and in particular donor hearts, below the freezing pointof water (0° C.) and recover them from freezing in a useful state, i.e.a state capable of maintaining coordinated cardiac contraction.Furthermore by using the solution according to the invention with suchadducts, it has been possible to reduce the temperature of intactmammalian subjects below the freezing point of water (0° C.) and restorethem from freezing in a state capable of maintaining coordinated cardiaccontraction and even respiration and conscious reaction. Other organsystems are also believed to be maintained with a high degree ofbiological integrity, i.e. in a physiological state capable ofmaintaining life.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the synthesis of the invention and is not intended to limitthe scope of what the inventors regard as their invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.), but some experimental error and deviationshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1. Solution Compositions

A. Composition of L solution. The composition of L solution is asfollows: Na⁺ 143 mM; Ca⁺⁺ 2.5 mM; Mg⁺⁺ 0.45 mM; K⁺ mM 3.0; Cl⁻ 124 mM;glucose 5 mM; and lactate 28 mM. The solution is filtered to removeundissolved material and placed in autoclavable containers and heated inan autoclave to a temperature of 120°C. for 15 minutes.

B. Composition of HL (BioTime Hextend™-lactate) Solution. L formulationwith the addition of 60 g/l of high molecular weight Hetastarch.

C. Composition of HLB (BioTime Hextend™-lactate-bicarbonate) Solution.HL solution with the addition of 5 ml/l 1 M solution of NaHCO₃.

D. Composition of HL-DL (BioTime Hetadex™-lactate) Solution. HLformulation except 6% Dextran 40 is used in place of 6% Hetastarch.

E. Composition of AL (BioTime Albextend™) Solution. L solution exceptwith the addition of 50 g/l albumin. ALB solution is AL solution withthe addition of 5 ml/l of a 1 M NaHCO₃.

F. Composition of HL-Heme Solution. HL solution with the addition of20-200 g/l hemoglobin.

G. Composition of L-Heme Solution. L solution with the addition of20-200 g/l hemoglobin.

EXAMPLE 2. Blood Replacement with HL-DL Solution

A 240 g female rat was anesthetized with ketamine, xylazine anacepromazine mixture injected i.m. The animal was placed on a stage andits right femoral artery and vein cannulated. The animal was perfusedisovolemically with 10 ml of HL-DL solution until its hematocrit reached17.2%. The cannulas were removed, vessels ligated, and the incisionclosed. The animal tolerated perfusion well, and was active and eatingwithin 3 days of the procedure. The animal recovered fully and remainedalive and healthy.

EXAMPLE 3. Reviving An Ice-Cold Blood-Substituted Dog

A 26.8 kg male dog was anesthetized with nembutal and intubated. It wasmoved to the operating room, ventilated, and catheterized with venous,Foley, arterial, and Swan-Ganz catheters, and after i.v. heparin, itsright femoral artery and vein were cannulated. An esophageal tube wasinserted and antacid administered. Temperature sensors were placed inthe esophagus and the rectum. Methyl prednisolone was injected i.v.

The animal was wrapped in a cooling blanket, and surface coolinginitiated. The animal's cannulas were connected to a bypass circuit,which consisted of a vortex blood pump, an oxygenator with a built-inheat exchanger, a secondary in-line heat exchanger, and a funnel for therapid administration of blood and blood substitute. Whole blood (225 ml)was removed from the dog and saved for rewarming. Blood volume wasquickly replaced with HLB solution. The bypass circuit containing 1.05liters of HLB solution was opened to the animal, and core cooling began.

Thirty three liters of HLB solution were exchanged. After substantiallyall of the blood was removed, a sufficient amount of a blood 2 M KClsolution was infused to stop cardiac contraction. By the time theice-point was approached, the hematocrit was far below 1%. The animal'sdeep esophageal temperature was below 10°C. for 4 hours and 5 minutes,with a minimum recorded temperature of 0.7°C.

Following the hypothermic period, the animal was warmed. When bodytemperature climbed past 10°C., venous effluent and whole bloodpreviously collected, as well as donor blood, was returned to thecircuit; hematocrit increased with increasing temperature. Lidocaine andbicarbonate were administered, the heart defibrillated, and ventilationbegun. When blood pressure and body temperatures approached normal, theanimal was weaned from bypass, and protamine and Lasix injected. Severalhours after warm-up, the animal was conscious and responsive. The animalremained alive and healthy after the procedure.

EXAMPLE 4. Reviving an Ice-Cold Blood-Substituted Baboon

A 24 kg male baboon of the species Papio annubis was anesthetized firstwith ketamine and acepromazine i.m., then with i.v. pentothal. It wasthen immobilized with pancuronium bromide. It was intubated, ventilated,and catheterized with venous, Foley, and arterial catheters. The animalwas wrapped in a cooling blanket, and surface cooling initiated. Afteri.v. heparin was administered, the baboon's right femoral artery andbilateral femoral veins were cannulated. Temperature sensors were placedin the esophagus, rectum and brain. The animal was instrumented for EKG,somatosensory evoked potentials (SSEPs) and EEG. Dexamethazone wasinjected i.v.

The animal's cannulas were connected to a bypass circuit, whichconsisted of a vortex blood pump, an oxygenator with a built-in heatexchanger, and a funnel for the rapid administration of blood and bloodsubstitute. Whole blood (300 ml) was removed from the baboon and savedfor rewarming. The volume was quickly replaced with 300 ml ofphysiological saline solution. The bypass circuit, containing 2 litersof Plasmalyte (commercially available electrolyte solution), was openedto the animal and core cooling begun.

After the deep esophageal temperature declined below 13°C., another 2liters of Plasmalyte containing 12.5 g of mannitol, was added to thecircuit, replacing the mixture of blood and Plasmalyte which previouslyfilled the circuit. This diluted blood was saved for use during warming.Immediately afterwards, 10 liters of HLB solution were added, replacingthe Plasmalyte. By the time the ice-point was reached, the hematocritwas far below 1%. When the animal reached brain temperature of 3.40°C.and deep esophageal temperature of 2.8°C., the blood pump was stoppedand the animal was maintained under a condition of circulatory arrest(standstill) for 45 minutes. After this period, circulation was resumed.

Following the hypothermic period, 4.2 liters of HLB solution wereflushed through the animal while collecting venous effluent, and theanimal warmed. When body temperature reached 150°C., 2 liters ofPlasmalyte were added to the circuit to replace the HLB solution.Mannitol (6.25 g/l) was added to the Plasmalyte in the circuit.Additionally, venous effluent and whole blood previously collected, aswell as donor blood cells and fresh-frozen plasma, were returned to thecircuit; the animal's hematocrit increased with increasing bodytemperature. Another 12.5 g of mannitol were added to the circuit. Whenthe esophageal and rectal temperatures approached normal, the heartfibrillated during warming and began beating. Ventilation was begun.When blood pressure and body temperatures approached normal, the animalwas injected with protamine i.v., weaned from bypass, its cannulas andcatheters removed, and its incisions closed.

The animal's deep esophageal temperature had been below 15°C. for 3hours, and below 10°C. for 2 hours 17 minutes, with a minimum recordedtemperature of 2.8°C. (Table 3). The following morning, the animal wasable to sit erect in its cage and pick up and eat pieces of banana, aswell as drink apple juice. It remained alive and well until sacrificedmore than one week later for histological evaluation.

EXAMPLE 5. Blood Replacement with Two Solution System in a PatientUndergoing Cardiopulmonary Bypass Surgery

A patient is anesthetized, cannulated and instrumented forcardiopulmonary bypass. The patient is wrapped in a cooling blanket andsurface cooled to 30°C. The patient is then placed on bypass with thecircuit primed with ALB solution. The patient is core and surface cooleduntil his deep esophageal temperature reaches 20°C. Blood is collectedwith 4 L of ALB solution, and immediately replaced with HLB solution.The body is then cooled and maintained while surgical procedures areperformed on the heart or brain. The patient is then warmed, and the HLBsolution replaced first with ALB solution, and then with the ALB-bloodsolution originally removed. 5-10 mg of vitamin K is administered.

One of the advantage of using the ALB solution as a bypass prime and forblood collection is that when the patient's own hemodiluted blood isreinfused during warm-up, albumin functions as the naturally-occurringcompound, maintaining blood oncotic agent without impeding the patient'sown ability to synthesize albumin.

EXAMPLE 6. Emergency Blood-Substitution of Hemorrhaging Subject withHL-Heme Solution

A patient suffering from severe blood loss is infused with HL solutioncontaining 5 mg/l of blood-clotting factor vitamin K and 30 g/l of theoxygen-carrying component hemoglobin. The patient's blood pressure isstabilized and normal oxygen delivery to the patient's tissues isresumed. The patient's body gradually clears the Hetastarch componentwhile synthesizing its own albumin such that blood oncotic pressureremains stabilized during the recovery period.

Use of HL solution containing blood-clotting factors and oxygen-carryingcomponents allows the use of substitute blood to be reduced orcompletely avoided.

EXAMPLE 7. Use of Blood Clotting Factor in Hemodiluted Mammals

Six young female rats (227-262 g) were anesthetized, their right femoralarteries and veins cannulated, and 40-60% of their blood replaced withHL solution. After hematocrits were reduced to 16-22%, the animals wereslowly injected i.v. with 6 ml of Trasylol® (10,000 KIU/ml). Their tailswere severed 30 mm above the tip. Blood loss averaged 0.39±0.06(mean±SEM) ml, and all but one animal survived at least one day. Eightcontrol animals were similarly perfused with HL solution, but were notgiven Trasylolg injections. The average blood loss was 4.8±0.54 ml, andonly 3 of the 8 animals survived. Compared to untreated controls,mortality (P<0.02) and blood loss (P<0.002) in the HL-treated animalswithout Trasylol® was significantly greater.

EXAMPLE 8. Ice-Cold Blood Substitution of a Dog with Solution HLB

Place a 25-30 Kg dog on partial cardiopulmonary bypass. Surface and corecool the dog to near the ice point (1-3° C.). Replace the dog's bloodwith solution HLB hypothermic blood substitute, described in Example 1.Retain the blood for transfusion during rewarming. Reduce the animal'sbody temperature to near the ice point (below 4° C.) and then rewarm.Replace the blood substitute with blood with warming and revive theanimal.

Preparation. Catheterize the dog by means of the right radial vein,injected iv with pentothal, then fit with an endotracheal tube andventilate with isofluorane (or Flether) in 100% O₂. Initiate a Ringer'slactate drip at a rate titrated to the dog's arterial blood pressure(approx. 40 ml/hr iv). Place the dog on a cooling blanket cooled withrecirculating ice water. Catheterize the right carotid artery to allowfor blood pressure (CAP) monitoring, and add a 3-way stopcock in-line toallow arterial blood sampling every 10-60 min. throughout the entireprocedure. Insert a foley catheter for urine collection and measure theurine volume throughout the procedure. Implant a 2 lumen, 7 F, Swan Ganzwedge catheter via the right jugular vein or right femoral vein, whichis fed through the right heart into the pulmonary artery. Use the distalport to measure pulmonary wedge pressure (PAW), the proximal port isused for central venous pressure (CVP). (If necessary CVP may bemeasured with a catheter inserted in one of the brachial veins.) Isolatethe left femoral artery and vein and prepare for cannulation. Heparinizethe animal (approx. 5,000 u). Insert a Biomedicus venous return cannula(15-19 F) in the femoral vein and a Biomedicus arterial cannula (12-15F) in the femoral artery. Measure the activated clotting time (ACT)every 45 min. (until blood substitution) and adjust the heparin suchthat it remains greater than 400 sec. Attach a thermocouple approx.midway to an esophageal tube and insert the unit so that the tube entersthe stomach. A second thermocouple is placed rectally.

Attach ECG leads. Add Solu-Delta-Cortef (Upjohn, veterinary prednisoloneNa succinate), 80 mg by iv injection. Coat the eyes with Terrimycin (orLacrylube), and add DiGel (or Maalox, 20 ml) through the esophagealtube.

Measurements. Measure arterial blood gasses, pH and hematocrit in everyblood sample, and in some cases electrolytes, enzymes and otherchemistries. Monitor esophageal and rectal temperature as well as thearterial inflow and venous return blood temperatures. Monitor CAP, PAW,CVP, ECG, and airway pressure. Temperatures should be displayeddigitally and stored as a function of time in a computerized dataacquisition system. The pressures and ECG should be displayed as realtime waveforms or as numerical data and stored by the computer.

Bypass Circuit Components. The circuit features a Biomedicus centrifugalblood pump and flow meter, a Terumo hollow fiber membrane oxygenatorwith built-in heat exchanger, Shiley hard shell venous reservoir withfilter and a secondary heat exchanger with integral bubble trap(Electromedics) located as close to the animal as possible. A drainsegment is located near the inlet of the venous reservoir and terminateswith a check valve. This allows rapid and efficientblood/blood-substitute exchanges. There is an A-V shunt segment thatallows circulation when not on bypass.

The venous reservoir can be filled from either the 1 liter separatoryfunnel through the “quick prime” port or from dual infusion bags throughone of the cardiotomy ports. The arterial line from the oxygenator tothe arterial cannula and the A-V shunt are constructed from ¼″ tubing;the venous return, drain and pump-head lines are ⅜″. In those segmentswhere severe bending can occur, heavy-wall tubing is used or the tube isbraced with “spiral wrap.”

The patient loop is double wrapped and the entire circuit (sans thefactory sterilized reservoir, secondary heat exchanger and oxygenator)is ethylene oxide gas sterilized as six basic sections (pump-head, flowmeter section, central bypass loop, funnel, infusion line, and gasfilter line).

Bypass Circuit Support. Ice water, pumped from one of two 10 gal.insulated reservoirs, is used to cool the oxygenator and secondary heatexchangers. The other reservoir supplies the cooling blanket. At theonset of surgery, ice water is circulated through the cooling blanket.At the onset of bypass, room temp. water is circulated through thecircuit heat exchangers.

Temperature is slowly decreased by adding ice to the reservoir, inquantities sufficient to maintain a 7-10°C. difference between theesophageal and blood stream temperatures. After blood substitution (i.e.to a hematocrit of less than about 4%) full ice water flow is commenced.

Upon rewarming, ice is removed from the reservoir and the heater isactivated. The temperature of the warming stream is limited to a maximumof 10°C. greater than the venous return temperature, by manualadjustment of the heater thermostat.

The oxygenator is supplied with sterile, filtered 100% O₂.

Blood Substitution. The circuit is primed with 2 liters of solution L(Example 1), and recirculated through the A-V shunt to ensuretemperature-gas equilibrium. The cannulas are attached to the arterialand venous lines of the bypass circuit, and the lines remain clamped.The cooling blanket is wrapped around the patient who is surface cooleduntil a deep esophageal temperature of 35° C. is reached.

The clamps are removed, and bypass is commenced with the solutionL-diluted blood stream at room temperature (approx. 25° C.). At theonset of cooling, gaseous anesthesia is discontinued, and the dog ismanaged with 2.5% pentothal.

The blood stream is gradually cooled until the animal has an esophagealtemperature of 20° C., at which time blood is removed by clamping thevenous return at the reservoir inlet and draining from the drain segmentwhile L solution is infused. During this exchange, an additional 2liters of L solution is added to the venous reservoir and when the levelof L solution drops to 250 ml, approximately 6 liters of HLB is addedstepwise until all of the blood is removed (HCT less than 2%, visualobservation). Approximately 4 liters of blood/blood-substitute mixturescollected in sterile bottles and retained for reinfusion. The verydilute blood mixture (about 5 ½ liters) is discarded.

After 4 liters have been exchanged (i.e. after the addition of 2 litersof solution L and 2 liters of solution HLB), 20 meq KCl will be injectedvia a stopcock on the secondary heat exchanger, to arrest the heart.During the exchange, the inflow is adjusted such that the PAW is keptbelow 5 mm Hg and the rate of efflux equals the rate of influx, i.e. asclose to isovolemia as possible. At the end of the exchange the finalreservoir level will be about 500 ml, the PAW below 5 mm Hg and the CVPless than 5 mm Hg. Flow will be adjusted such that isovolemia will bemaintained (constant reservoir level and the above pressure levels, i.e.PAW<5 mm Hg and CVP<5 mm Hg).

When almost all of the blood is removed (HCT less than 4%, visualobservation), the cooling stream can be reduced to ice water temperature(filling the reservoir with ice), and the dog rapidly cooled to itsminimum temperature. If the HCT is observed to rise at any time duringcold perfusion, the blood mixture can be removed by exchanging with 2 to4 liters of solution HLB by the method described above.

During the entire procedure, arterial blood samples are taken and bloodgasses, pH, HCT, and in some cases electrolytes, and other bloodchemistries monitored.

After about 1-2 hours of blood substituted cooling, the dog'stemperature will be about 1-4° C., and rewarming will begin. The dogwill be rewarmed, by removing the ice from the supply reservoir andwarming its contents with the heater which in turn warms the blankets.When the esophageal temp reaches 15° C., 4 liters of solution L with 25g mannitol will be exchanged with the solution HLB followed by the 4liters of collected blood mixture. The effluent will be discarded.

The animal will be transfused with stored autologous or donor blood andwarmed gently with a warming temperature differential less than 10°C.and never above 40° C. The heart will spontaneously begin to beat orwill be difibrillated. When the animal's temperatures (esophageal andrectal) reach about 35° C., physiological parameters are stabilized, andit can support itself, it can be weaned from the extracorporeal circuit.

EXAMPLE 9. Concentrated aqueous solution of magnesium and potassium forcardio-protection

The discovery disclosed here is a novel product, specifically, anaqueous solution consisting of high concentrations of K⁺ and Mg²⁺ to beused in conjunction with ice cold HLB blood substitution in subjectsrequiring low-temperature surgery, in subjects requiring cardioplegia,or in organ donors to better preserve organs—such as hearts—prior totransplantation.

This solution can be used in conjunction with hypothermic total bodywashout with the HLB solution at cold temperatures to provide bettercardiac protection during periods of circulatory arrest. The solutioncan also be used as a cardioplegia agent along with HLB in standardcardiac surgery procedures not involving total body washout, or topreserve the heart for transplantation after total body washout withcold HLB solution.

In preparing the high concentration solution of Mg and K, MgSO₄ and KClhave been used. These compounds are currently available in a formapproved for use in human patients by the US FDA. However, MgCl₂ maysubstitute for MgSO₄ and KOH may also be useful in providing a basic pHat about 7.8 (range 7.2-8.4). Other substances which may be used inpreparation of the high concentration solution of K and Mg are KHCO₃,K₂PO₄, K lactobionate, Kcitrate, Kacetate and Kgluconate, as well as theaddition of NaHCO₃ to the solution containing K⁺ and Mg⁺⁺. The endresult however being a solution containing approximately 1.5 M potassiumand 0.5 M magnesium.

Initially, a solution containing 50 mM KCl and 10 mM MgSO₄ in HLBsolution. was prepared. Approximately 1 to 2 ml of this solution wasadministered iv or intra-arterially to induce cardiac arrest of a smallice-cold blood substituted hamster. This volume corresponds to about30-50% of a blood hamster's blood volume. It was discoveredexperimentally that this solution, when bolused intravascularly justupon completion of total body washout, induces cardiac arrest.

It was found that when about 9.5 ml of a 50% solution of MgSO₄.7H₂0(i.e. approx. 2M) and 27 ml 2M KCl was administered intra-arteriallyinto ice cold dogs (body weights about 25 kg, with estimated bloodvolumes of about 1.8 liters) whose blood was replaced with HLB,significantly better protection of the heart resulted . compared to thatwhen no additional K and Mg is administered or when a more diluted K/Mgsolution is administered (i.e. first dissolving the concentrated K/Mgsolution in approximately 500 ml HLB solution in an oxygenatorreservoir, and further diluting the solution in a circuit containing anadditional 850 ml of HLB, which would create a 6.2 mM Mg⁺⁺ and 17.4 mMK⁺ solution).

The solution, containing about 37 ml of 0.55M MgSO₄.7H₂0 and 1.48M KCl,was adminstered into a component of the bypass circuit (heat exchanger)which was directly connected to the femoral artery of the subject. Basedon this dog experimentation, this solution is expected to provideimproved cardiac protection in subjects whose blood is replaced with HLBat low temperature.

Similarly, when delivering 0.1 ml of a high concentration Mg⁺⁺/K⁺ mixintra-arterially into hamsters, it effectively provides cardiacprotection when used just after hypothermic total body washout with HLB.

The novel discovery described here is the use of a solution consistingof extremely high concentrations of Mg and K which can be administeredsafely and effectively to induce cardioprotection particularly inconjunction with cold subjects whose blood has been replaced with HLB.The concentration of K⁺ and Mg⁺⁺ administered is more than 10 timeshigher (for both electrolytes) than previously used or described toprotect cold hearts.

1. Revival of a hamster using HLB and concentrated Mg/K solution.

A 55 g female hamster, fasted overnight was anesthetized with Ketamine0.03ml (100 mg/ml solution) and covered with crushed ice to lower rectaltemperature to about 13° C. The animal was then placed on a surgicalplatform under a stereo microscope. Cannulas were placed in the carotidartery and jugular vein. The HLB solution was perfused into the arterywhile venous effluent was collected. The blood was replaced withice-cold HLB solution. When about 65% of the blood was removed, theanimal's temperature was lowered to about 1° C. Ventilation with 100% O₂was initiated when the animal's temperature fell below 10°C.Approximately 7 ml or 2 blood volumes was required to replace most ofthe circulating blood. Upon cessation of perfusion, a 0.2 ml bolus(I.V.) of 0.5M KCl with 0.1 M MgSO₄ was given. The heart arrestedimmediately. The animal was maintained in cardiac arrest for 5 hrs and30 min. The animal was then perfused with 3 ml HLB followed by bloodtaken from donor animals. Upon gradual rewarming, normal heart beat andbreathing resumed. On recovery, the animal demonstrated an ability torespond to stimuli, such as gentle pressure on the abdomen or paws.

2. A 70g hamster, fasted overnight, was anesthetized with 0.03 mlketamine (100 mg/ml solution) and covered with crushed ice to lowerrectal temperature to about 13C. The animal was then placed on asurgical platform under a stereo microscope. Cannulas were placed in thecarotid artery and jugular vein. The HLB (with added glucose 50 mM and 5mM NaHCO₃) solution is perfused into the artery while venous effluent iscollected. The blood was replaced with ice-cold HLB solution. When about65% of the blood was removed the animals temperature was lowered toabout 1 C. Ventilation with 100% O2 was initiated when the animalstemperature fell below 10 C. Approximately, 7 ml or 2 blood volumes isrequired to replace most available blood.

A solution consisting of 0.02 ml 1 M MgSO₄ plus 0.1 ml KCl 1 M isdiluted with 0.1 ml HLB. Then the final 0.22 ml solution is administerediv. This corresponds to delivery of a cocktail 0.5M KCl and 0.09M MgSO₄.After 5 hours of circulatory arrest the animal is perfused with 3 ml HLBthen blood taken from donor hamsters. As the animal is warmed, heartbeat, breathing, and responsivity resume.

Previous to the discovery of using the Mg/K concentrate it was notpossible to repeatedly obtain such results with regard to revival ofhamster after 5 hours of cardiac arrest.

3. A 70g female hamster, fasted overnight, was anesthetized withKetamine 0.03 ml (100 mg/ml solution) and covered with crushed ice tolower rectal temperature to about 13C. The animal was then placed on asurgical platform under a stereo microscope. Cannulas were placed in thefemoral artery and jugular vein. The HLB solution is perfused into theartery while venous effluent is collected. The blood was replaced withice-cold HLB solution. When about 65% of the blood was removed theanimals temperature was lowered to about 1C. Ventilation with 100% O2was initiated when the animals temperature fell below 10C.Approximately, 5 ml was infused. Then 0.2 ml of a cocktail 0.5 M KCl and0.09M MgSO₄ is administered intra-arterially. Then 1 ml of HLB wasinjected to chase the “cardioprotectant” up the circulatory system andinto the tissues. The animal was maintained in circulatory arrest for 1hour. Then the animal was perfused with 4 ml HLB and blood from donorswhile being rewarmed gradually. It was observed the hamster's heartresumed a normal EKG signal, followed by spontaneous breathing,responsiveness, consciousness, upright posture and long term survival.

Previously, long term survival was only rarely accomplished in suchexperiments without use of the K/Mg concentrate.

EXAMPLE 10. Method for Using HLB Involving Intermittent Perfusion withAdditional Bicarbonate

During periods of ice-cold blood substitution and circulatory arrest itwas found that certain neurologic recovery takes longer compared tocontinuous circulation of HLB solution. In experiments on hamstersimproved results were obtained using both extra bicarbonate in HLBand/or intermittent perfusion with HLB to reduce acidosis. This alsoprevents rigor and improves recovery especially of brain function asnoted by responsivity following long periods of cardiac arrest inhamsters.

It has also been found that slow continuous perfusion of cold HLB bloodsubstituted dogs appears to provide longer periods of cardiac arresttime than without continuous perfusion.

In hamsters it was found that additional bicarbonate, 5 mM, can behelpful at reducing acidosis after long periods of circulatory arrest.

1. A 60g hamster, fasted, was anesthetized with Ketamine 0.03 ml (100mg/ml solution) and covered with crushed ice to lower rectal temperatureto about 13° C. The animal was then placed on a surgical platform undera stereo microscope. Cannulas were placed in the carotid artery andjugular vein. HLB solution (with added glucose 50 mM and 5 mM NaHCO₃)was perfused into the artery while venous effluent was collected. Whenabout 65% of the blood was removed the animals temperature was loweredto about 1° C. Ventilation with 100% O₂ was initiated when the animalstemperature fell below 10°C. Approximately, 7 ml or about 2 bloodvolumes is required to replace most available blood. The additional 5 mMNaHCO₃ provided a final pH of 8.5. After perfusion, the heart wasstopped with 0.5 ml of 50 m MKCl and 10 mM MgSO₄. After 4 hours and 40minutes the animal was re-perfused with 2 ml HLB followed by perfusionof blood taken from donor animals. The animal did not recover tobreathing and responsiveness.

2. The same experiment was performed as in 1 above except that anintermittent perfusion of bicarbonate was introduced for 15 minutes 2hours and 30 minutes after circulatory arrest.

A 60g hamster, fasted, was anesthetized with Ketamine 0.03 ml (100 mg/mlsolution) and covered with crushed ice to lower rectal temperature toabout 13° C. The animal was then placed on a surgical platform under astereo microscope. Cannulas were placed in the carotid artery andjugular vein. The HLB (with added glucose 50 mM and 5 mM NaHCO₃)solution was perfused into the artery while venous effluent wascollected. The blood was replaced with ice-cold HLB solution. When about65% of the blood was removed the animals temperature was lowered toabout 1°C. Ventilation with 100% O₂ was initiated when the animalstemperature fell below 10°C. Approximately 7 ml or 2 blood volumes wasrequired to replace most available blood. Perfused HLB (containing anadditional 50 mM glucose) plus an additional 5 mM NaHCO₃ which provideda final pH of 8.5. After perfusion, the heart was stopped with 0.5 ml of50 mM KCl and 10 mM MgSO₄. After 2 hours and 30 minutes, the animal wasperfused with HLB for 15 minutes (i.e. 2 ml HLB). After 4 hours and 40min re-perfused with 2 ml HLB followed by blood from donor animals andwarmed. The animal revived to breathing and responsivity. The time ofcardiac arrest was over 5 hours.

EXAMPLE 11. Cryoprotective Solutions

1. Preparation of solution

a. Method 1

To prepare 50 ml of a 10% glycerol solution in HLB, 0.45 ml of HLB ispoured into a tube. Then 5 ml of 100% glycerol is added. The solution isshaken and filtered through 0.2 micron filter or less.

b. Method 2

For 1 liter HLB 15% glycerol: Add HES 60 g/l; NaCl 6.72*g/l bring volumeto ½ final volume i.e. 500 ml with de-ionized water. Add and dissolve byshaking one chemical at a time MgCl₂ 0.09 g/l; CaCl₂ 0.37 g/l; KCl 0.22g/l; glucose 0.9 g/l; Na Lactate (60% syrup) 4.03 ml/L; NaHCO₃ 0.42 g/lor 0.84 g/l (the additional bicarbonate may help reduce acidity). Bringto 850 ml with de-ionized water. Add 150 ml 100% glycerol. Filter 0.2u * the amount of NaCl added is adjusted upon accounting for NaCl instarch note: with 50 mM extra glucose add 9 g/l additional glucose withshaking

1. A cryoprotective solution of 10% glycerol in HLB allows revival ofpartially frozen hamsters.

A 60g female hamster, fasted overnight was anesthetized with Ketamine0.03 ml (100 mg/ml solution) and covered with crushed ice to lowerrectal temperature to about 13°C. The animal was then placed on asurgical platform under a stereo microscope. Cannulas were placed in thecarotid artery and jugular vein. The blood was replaced with 2 mlice-cold HLB solution followed by cryoprotective solution of HLB plus10% glycerol. When about 65% of the blood was removed the animalstemperature was lowered to about 1° C. Ventilation with 100% O₂ wasinitiated when the animals temperature fell below 10°C. After perfusionof 5 ml of cryoprotective solution, the heart was arrested with 50 mMKCland 10 mM MgSO₄ (1.5 ml i.v.). Animals were then placed in a plastic bagand immersed in a cooling bath set at −15° C. After 30 minutes rectaltemperatures dropped as cold as −2.2° C.

The animal was thawed, reperfused with HLB and then with donor blood.Upon rewarming, normal EKG signals are observed followed by breathingand responsiveness. Massive and extensive lesions (micro-hemorrhages)were observed in the brain upon necropsy demonstrating that the brainwas substantially frozen under these conditions. However, despitefreezing animals can recover to responsiveness.

2. Cryoprotective solution of 10% glycerol and 10% DMSO

A 60g female hamster, fasted overnight was anesthetized with Ketamine0.03 ml (100 mg/ml solution) and covered with crushed ice to lowerrectal temperature to about 13° C. The animal was then placed on asurgical platform under a stereo microscope. Cannulas were placed in thecarotid artery and jugular vein. The blood was replaced with 1.5 mlice-cold HLB solution followed by cryoprotective solution of HLB plus10% glycerol and 10% DMSO. When about 65% of the blood was removed theanimals temperature was lowered to about 1° C. Ventilation with 100% O₂was initiated when the animals temperature fell below 10°C. After about6.5-7.0 ml of cryoprotective solution was perfused and the heart wasarrested with 50 mM KCl and 10 mM MgSO₄ (1.5 ml i.v.) The animals wereplaced in a plastic bag and immersed in a cooling bath set at −15° C.After 30 minutes rectal temperatures dropped as cold as −1.5° C.

Upon thawing and reperfusion with HLB followed by donor blood, normalheart beat, breathing and responsiveness resumed. Massive and extensivelesions were observed in the brain upon necropsy demonstrating that thebrain was substantially frozen under these conditions and that despitefreezing animals can recover to responsiveness.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A physiologically acceptable aqueous solutioncomprising: electrolytes; a dynamic buffering system comprising acarboxylic acid, salt or ester thereof wherein said carboxylic acid,salt, or ester thereof is selected from the group consisting of lactate,acetate, gluconate, citrate and pyruvate; and at least one oncoticagent, wherein said at least one oncotic agent is selected fromhydroxoethyl starch magnesium; and a simple sugar in an amount of notmore than about 50 mM; and further wherein said solution does notcomprise a biological buffer.
 2. A physiologically acceptable aqueoussolution comprising: electrolytes; a dynamic buffering system comprisinga carboxylic acid, salt or ester thereof, wherein said carboxylic acid,salt or ester thereof is selected from the group consisting of lactate,acetate, gluconate, citrate and pyruvate; a simple sugar in an amount ofnot more than about 50 mM; and at least one oncotic agent, wherein saidat least one oncotic agent is a medium or high molecular weighthydroxyethyl starch; wherein said solution does not comprise abiological buffer.
 3. The solution according to claim 1, wherein saiddynamic buffering system further comprises bicarbonate.
 4. The solutionaccording claim 1, wherein said solution further comprises acryoprotective agent.
 5. The solution according to claim 1, wherein saidhydroxyethyl starch is a medium molecular weight hydroxyethyl starch. 6.The solution according to claim 1, wherein said hydroxyethyl starch is ahigh molecular weight hydroxyethyl starch.
 7. The solution according toclaim 1, where said electrolytes comprise sodium, potassium, calcium,chloride ion and magnesium.
 8. The solution according to claim 1,wherein said solution further comprises a clotting factor.
 9. In amethod where an aqueous composition is introduced into the circulatorysystem of a host or portion thereof, the improvement comprising usingthe physiologically acceptable aqueous solution according to claim 1 asthe plasma substitute solution.